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Literature summary extracted from

  • Zheng, Z.; Sheng, B.; Gao, C.; Zhang, H.; Qin, T.; Ma, C.; Xu, P.
    Highly stereoselective biosynthesis of (R)-alpha-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase (2013), Sci. Rep., 3, 3401.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.28 gene ldhD, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Lactobacillus delbrueckii subsp. bulgaricus

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.28 F299Y site-directed mutagenesis Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 additional information highly stereoselective biosynthesis of (R)-alpha-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase, asymmetric reduction of a homologous series of alpha-keto carboxylic acids such as phenylpyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid, beta-hydroxypyruvate, overview. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH shows elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-alpha-hydroxy carboxylic acids can be produced at high yields and highly optical purity. Production of chiral (R)-phenyllactic acid. 50 mM PPA is completely reduced to (R)-phenyllactate in 90 min with a high yield of 99.0% and a highly optical purity (99.9% e.e.) by the engineered coupled production system. Activties of the F299Y mutant are similar to the wild-type enzyme Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 Y52L site-directed mutagenesis Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 Y52L/F299Y site-directed mutagenesis Lactobacillus delbrueckii subsp. bulgaricus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.28 0.27
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant Y52L Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 0.32
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant F299Y Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 1.4
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 11.4
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged wild-type enzyme Lactobacillus delbrueckii subsp. bulgaricus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.28 (R)-lactate + NAD+ Lactobacillus delbrueckii subsp. bulgaricus
-
pyruvate + NADH + H+
-
r
1.1.1.28 (R)-lactate + NAD+ Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842
-
pyruvate + NADH + H+
-
r
1.1.1.28 pyruvate + NADH + H+ Lactobacillus delbrueckii subsp. bulgaricus
-
(R)-lactate + NAD+
-
r
1.1.1.28 pyruvate + NADH + H+ Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842
-
(R)-lactate + NAD+
-
r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.28 Lactobacillus delbrueckii subsp. bulgaricus
-
-
-
1.1.1.28 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.28 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) to homogeneity by nickel affinity chromatgraphy Lactobacillus delbrueckii subsp. bulgaricus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.28 (R)-lactate + NAD+
-
Lactobacillus delbrueckii subsp. bulgaricus pyruvate + NADH + H+
-
r
1.1.1.28 (R)-lactate + NAD+
-
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 pyruvate + NADH + H+
-
r
1.1.1.28 2-oxobutyrate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus 2-hydroxybutyrate + NAD+
-
?
1.1.1.28 2-oxobutyrate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 2-hydroxybutyrate + NAD+
-
?
1.1.1.28 2-oxovalerate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus 2-hydroxyvalerate + NAD+
-
?
1.1.1.28 2-oxovalerate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 2-hydroxyvalerate + NAD+
-
?
1.1.1.28 additional information residues Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of 2-oxocarboxylic acids and their steric orientation in the wild-type enzyme. Substrate specificity and enantioselectivity of D-nLDH and DnLDH mutants, overview Lactobacillus delbrueckii subsp. bulgaricus ?
-
?
1.1.1.28 additional information residues Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of 2-oxocarboxylic acids and their steric orientation in the wild-type enzyme. Substrate specificity and enantioselectivity of D-nLDH and DnLDH mutants, overview Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 ?
-
?
1.1.1.28 phenylpyruvate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus D-phenyllactate + NAD+
-
?
1.1.1.28 pyruvate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus (R)-lactate + NAD+
-
r
1.1.1.28 pyruvate + NADH + H+
-
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (R)-lactate + NAD+
-
r

Synonyms

EC Number Synonyms Comment Organism
1.1.1.28 D-nLDH
-
Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 NAD-dependent D-lactate dehydrogenase
-
Lactobacillus delbrueckii subsp. bulgaricus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.28 37
-
assay at, 2-oxoacid reduction activity Lactobacillus delbrueckii subsp. bulgaricus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.28 1.8
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant F299Y Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 11.3
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged wild-type enzyme Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 1447
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 2013
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant Y52L Lactobacillus delbrueckii subsp. bulgaricus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.28 7.5
-
assay at, 2-oxoacid reduction activity Lactobacillus delbrueckii subsp. bulgaricus

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.28 NAD+
-
Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 NADH
-
Lactobacillus delbrueckii subsp. bulgaricus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.28 1
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged wild-type enzyme Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 5.7
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant F299Y Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 1000
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.28 7500
-
phenylpyruvate pH 7.4, 37°C, recombinant His-taged mutant Y52L Lactobacillus delbrueckii subsp. bulgaricus